Research into the mechanism of inflammatory pain led to the discovery of the biochemical pathway associated with inflammation. One such example of chronic inflammatory pain is osteoarthritis. The elucidation of this pathway led to the association of the pro-inflammatory prostaglandins, produced by the cyclooxegenase enzyme, with pain and inflammation. The first generation of anti-inflammatory pain relievers, were classified as non-steroidal anti-inflammatory drugs or NSAIDs. Common NSAIDs such as aspirin, ibuprofen, naproxen, and indomethacin inhibit the cyclooxygenase enzyme, and thereby reduce inflammation by lowering the production of prostaglandin E-2. Many new NSAIDs were developed over the last 30 years, most of which are available by prescription only.
Until the emergence of the discovery of a second form of the cyclooxygenase enzyme, now called COX-2, there had been no distinction made between the various NSAIDs, in terms of the mechanism of action, and their effect, Only the magnitude of pain relief, or the potency for inhibiting the COX enzyme was considered important. However, side effects from the use of NSAIDs by patients who suffer from chronic inflammatory pain began to emerge. The principle side-effect was gastrointestinal toxicity, and it manifested in the form of gastric erosion, or erosion of the mucosal protective lining of the stomach. By the early 90s, as the incidence of osteoarthritis and rheumatoid arthritis increased, this side-effect became significant, leading to over 16,500 deaths per year in the United States alone. A review article by Wolf, M et al., Gastrointestinal Toxicity of Nonsteroidal Antiinflammatory Drugs, The New England Journal of Medicine, Vol. 340, No. 24, 1888–1899 (1999), is hereby incorporated by reference in its entirety. According to this article, 13 of every 1000 patients with rheumatoid arthritis who take NSAIDs for one year have a serious gastrointestinal complication. According to data from the National Center for Health Statistics and the Arthritis, Rheumatism, and Aging Medical Information System, yearly deaths from NSIAD toxicity (1997) in patients suffering from rheumatoid arthritis or osteoarthritis constitute the 15th leading cause of death in America. This figure is similar to mortality from AIDS (16,685) and only slightly less than deaths from Leukemia (20,197), but considerably greater than the number of deaths from multiple myeloma, asthma, cervical cancer, or Hodgkin's disease.
While most NSAIDs are more selective for the COX-1 form of the enzyme, they also inhibit the COX-2 form to varying degrees. Some NSAIDs, such as indomethacin, reduce both COX-1 and COX-2 to the same degree. Surprisingly, NSAIDs can also induce or up-regulate COX-2.
The potency of NSAIDs to cause gastric erosion and rapidly induce COX-2 can be illustrated by observing data from animal studies in which COX-2 was induced in the rat stomach within 1 hour of administration of aspirin or indomethacin. Both short term and long term administration of NSAIDs have produced gastric erosion as verified by endoscopy studies. Long term studies are defined as NSAID ingestion for at least 3 months, but usually are done over 3–6 months.
In the late 90s, a new class of prescription drugs emerged termed the COX-2 inhibitors. The first two compounds in this class approved by the U.S. FDA were celecoxib and rofecoxib. These drugs inhibited COX-2 with little or no effect on COX-1, and were sufficiently potent to produce equivalent pain relief to other NSIADs. While these compounds were no more effective than the NSAID pain relievers, chronic use resulted in virtually little gastrointestinal toxicity.
COX-2, or cyclooxygenase-2 inhibitors inhibit cyclooxygenase and reduce prostaglandins without producing the degree of gastric erosion associated with NSAID drugs such as aspirin. A COX-2 inhibitor selectively inhibits the COX-2 form of the enzyme more than the COX-1 form. To be classified as a good COX-2 inhibitor, a compound should inhibit COX-2 at least five times more than COX-1, or should have at least a 5:1 ratio of COX-2 to COX-1. Preferably, a COX-2 inhibitor should have an even greater selectivity than 5:1 for inhibiting COX-2, or from 5:1 to 100:1. A good COX-2 inhibitor would be capable of producing a concentration level in the blood that would reduce pain by 80 to 90% by inhibiting COX-2, with little or no effect on the COX-1 form of the enzyme. The terminology for quantifying the potency of a cyclooxygenase-2 inhibitor is the Inhibitory Concentration that produces a reduction of prostaglandin E-2 by 50%, termed the IC50. An even better index is the Inhibitory Concentration that produces an 80% reduction. This is called the IC80. For purposes of this application, the term IC80 shall refer to the concentration of the compound that produced an 80% reduction in the principle pro-inflammatory cytokine or prostaglandin, PGE-2. Conversely, the concentration of the compound capable of producing an 80% inhibition of the COX-2 enzyme could also be referred to as the IC80. Likewise, the IC50 shall mean the concentration of the compound that produces a 50% reduction in PGE-2, or a 50% reduction in the activity of the COX-2 enzyme.
In-vitro testing or screening of COX-2 inhibitors can be conducted by measuring the inhibition of prostaglandin E-2, a pro-inflammatory prostaglandin, in human whole blood. This results in the calculation of the IC50 values, or the amount or concentration of the compound needed to inhibit COX-2 by 50%, or the IC80 value, the concentration of the compound necessary to reduce prostaglandin E-2 by 80%. This testing model measures the production of prostaglandin E2 (PGE2) by the COX-2 enzyme related pathways, when stimulated by LPS or some other inducer of the COX-2 enzyme. COX-1 activity is also measured by measuring the production of thromboxane (T×B2). Such assays are now considered to represent a more complete in-vitro picture of COX-2/COX-1 selectivity and potency.
An international group of scientists published a consensus review related to COX-2 screening assays in: Brooks et al; Interpreting the clinical significance of the differential inhibition of cyclooxygenase-1 and cyclooxygenase-2, Rheumatology 1999; 38: 779–788. In this consensus paper, the committee stated that the Human Whole Blood Assay developed by Patrignani et al (J Pharmacol Exp Ther 1994; 271: 1705–12) was the best assay available for assessing inhibition of COX-1 and COX-2, or evaluating new COX inhibitors. More recently, the William Harvey Modified Human Whole Blood Assay was developed as an extension of the original whole blood assay, and most of the NSAID drugs, as well as the newer COX-2 inhibitors have been screened using this method.
To determine the COX-2/COX-1 inhibitory activity according to the invention the William Harvey Modified Human Whole Blood/Cell Assay (WHMA) is used, as set forth in T. D. Warner et al., Nonsteroid drug selectivities for cyclo-oxygenase-1 rather than cyclo-oxygenase-2 are associated with human gastrointestinal toxicity: A full in vitro analysis, Proc. Natl. Sci. USA 96:7563–68 (1999), hereby incorporated by reference in its entirety. The results from this assay are used to calculate the IC50 and IC80-WHMA COX-2/COX-1 ratio, which is simply the numerical ratio of the COX-2 IC50 concentration divided by the COX-1 IC50 concentration, obtained using the WHMA. In addition, the potency of the compound for reducing or inhibiting COX-2 is thereby determined. This is done by measuring the inhibition of the two isoforms of the enzyme at different concentrations of the inhibitor, starting at very low concentrations, and increasing in a log fashion until at least an 80% inhibition is produced. This results in a log graph of the concentration versus inhibition curve, or a dose response curve.
Numerous studies have shown that the relative incidence of GI side effects from NSAIDs can be correlated to the relative COX-2 specificity of these anti-inflammatory agents. The higher the specificity for COX-2 over COX-1, the lower the incidence of GI upsets. Accordingly, cyclooxygenase inhibiting agents with increased COX-2 specificity may provide improved anti-inflammatory compositions having less incidences of gastrointestinal distress or side effects. It is becoming increasingly apparent that the gastric damage that can be caused by NSAIDs is not just related to their effect on COX-1. Dual suppression of COX-1 and COX-2 seems to be necessary for damage to occur (Wallace, J L et al., NSAID-Induced Gastric Damage in the rat: Requirement for Inhibition of Both Cyclooxygenase-1 and Cyclooxygenase-2. Gastroenterology, 2000; 119:706–14). Furthermore, selective inhibition of COX-1, which greatly reduced prostaglandin synthesis, did not produce gastric damage in the same study. On the other hand, selective inhibition of COX-2 did not appear to have any effect on gastric prostaglandin synthesis, and did not produce gastric damage.
Interestingly, when both COX-1 and COX-2 were inhibited, gastric damage was consistently observed. This, and other research, is providing a clearer picture of the relationship between COX-1, COX-2, and gastric erosion. It now appears that combined inhibition of COX-1 and COX-2 contribute to the side-effects, but more highly selective inhibition of either COX-1 or COX-2 alone, is not responsible.
However, too much selectivity for COX-2 over COX-1 may not be desirable for other reasons. Certain side-effects may result from COX inhibitors that are extremely selective for COX-2. For example, the cardiovascular benefit of aspirin, a predominantly COX-1 non-steroidal anti-inflammatory drug (NSAID), is thought to be due to its activity as an anti-platelet aggregating drug. COX-2 inhibition does not result in anti-platelet aggregation. Current pharmaceutical COX-2 inhibitors, such as celecoxib or rofecoxib, are highly specific COX-2 inhibitors, and would not be expected to have any COX-1 inhibitory activity at the doses used to reduce pain and inhibit COX-2 activity. Thus, the cardiac-related side effects that have been noted with the use of some COX-2 specific inhibitors may be related to the lack of any COX-l inhibition while significantly inhibiting COX-2.
Furthermore, an additional problem associated with highly specific COX-2 inhibitors is the increase in gastric erosion produced by concurrent administration with other non-steroidal anti-inflammatory drugs (NSAIDS). For example, if a patient is taking a highly selective COX-2 inhibitor and also takes aspirin for cardiovascular benefit, the aspirin will cause even worse damage to the gastric mucosa. The reason for this is that some of the prostaglandins that are inhibited by cyclooxygenase inhibitors, such as prostaglandin E-2 (PGE2), are protective of the gastric mucosa, and actually contribute to healing of ulceration. Low dose aspirin produces small erosions in the stomach, and at the site of these ulcerations, the COX-2 enzyme becomes up-regulated. When COX-2 is blocked by selective COX-2 inhibitors, the protection afforded by the beneficial prostaglandins is eliminated. The result is that the ulcerative damage is made even worse. Concomitant administration of selective COX-2 inhibitors with aspirin is therefore contraindicated. This phenomenon is an indication of the problems associated with the dual inhibition of both COX-1 and COX-2. Thus gastric erosion will be worse with a single compound that exhibits significant inhibition of both COX-1 and COX-2, or by combining a COX-2 selective compound with a non-selective COX inhibitor that also inhibits COX-1 to a large degree. The key to overall risk reward benefits would be to have just the right amount of COX-1 inhibition along with predominantly COX-2 inhibition. The ratio of IC50COX-2/IC50COX-1 would be from about 1:5 to 1:100 or numerically from 0.20 to 0.010. Preferably, the ratio of IC50COX-2/IC50COX-1 would be at least 20:1 or numerically 0.05. For example, a compound that is tested using the WHMA protocol might have an IC50 for COX-2 of 1 μg/ml and an IC50 for COX-1 of 20 μg/ml, therefor, the IC50COX-2/IC50COX-1 ratio would be 1:20 or 0.05.
In summary, highly selective single entity COX-2 inhibitors such as rofecoxib and celecoxib, while important new drugs for the treatment of pain associated with osteoarthritis and other maladies, have some serious potential side-effects. These side effects can be divided into two major groups; 1) cardiovascular, and 2) worsening of gastric erosion when taken with aspirin or other NSAIDS. Both of these side effects may be related to an unbalanced total inhibition of the COX enzyme, and therefor, virtually complete blocking of prostaglandin production. Because prostaglandins have both positive and negative functions in the body, their total inhibition is a double-edged sword. Furthermore, there is a significant overlap in the patient populations that take both aspirin for cardiovascular benefit, and a selective COX-2 inhibitor for pain. Most of these subjects primarily consist of the elderly population. There is a significant need for anti-inflammatory pain relief without the negative side effects of the NSAIDs or the selective COX-2 inhibitors. Such a composition would provide pain relief while also inhibiting platelet aggregation, and providing protection for the gastric mucosa through some gastroprotective or cytoprotective mechanism. These second generation COX-2 inhibitors would be selective enough to inhibit COX-2 over COX-1, but not so selective that they would result in the additional side effects mentioned above. These compounds may exhibit protective activity by virtue of the existence of some other beneficial properties.
In the search for new anti-inflammatory compounds, many potential candidates have come from the plant kingdom. These botanicals are usually extracted and tested in-vitro for COX inhibition using various cell lines and methods. Usually these methods involve screening the compounds for COX-2 and COX-1 inhibition by measuring the inhibition of prostaglandin E-2 for COX-2 inhibition, and T×B2 for COX-1 inhibition. Selectivity can then be determined by calculating the COX-2/COX-1 ratio, or conversely, the COX-1/COX-2 ratio.
It would be desirable to find compounds that exhibit good selective COX-2 inhibition, but with the least amount of cardiovascular or gastrointestinal side-effects. Such compounds would result in a broader spectrum of therapeutic benefit, and be tunable over a wide range of COX-2/COX-1 ratios, providing effective pain relief with less side-effects. Such compounds could provide some minimal amount of COX-1 inhibition for cardiovascular benefit, without significant gastric erosion, while providing significant COX-2 inhibition for pain.
What are needed are compositions and methods that address the problems noted above.